Recent studies have implicated oxidative DNA damage in mediating neuronal death in cerebral ischemia and in neurodegenerative diseases. Little is known about how neuronal DNA repair mechanisms respond to ischemic injury and oxidative DNA damage. The broad, long-term objective of this project is to identify the role of inducible DNA excision repair in the regulation of cell death from acute ischemic brain injury. The specific objectives are: (1) Determine inducible DNA excision repair activity in an in vivo rat model of focal ischemia and reperfusion. (2) Determine inducible DNA excision repair activity in mechanist-ically defined in vitro models of neuronal ischemia and neuronal cell death. (3) Determine activity of selected DNA repair enzymes and expression of DNA repair genes in vivo focal cerebral ischemia and in vitro cell culture models of ischemia and neuronal cell death. (4) Determine the role of selective DNA repair enzymes in inducible DNA excision repair in focal ischemia and in vitro models of ischemia and neuronal death, by altering the activity of these enzymes. in vitro models allow for precise mechanistic studies to be performed, but the complex inter-dependent pathophysiologic changes that occur after ischemia in vivo cannot be completely modeled. Accordingly, both primary cortical neuronal cultures and rat temporary middle cerebral artery occlusion models will be used. Methods for detection of single, double and total DNA breaks will be used to study the evolution of DNA damage in these models. The ability of whole-cell extracts to repair damage to plasmid DNA by base excision repair or nucleotide excision repair will be used to assay DNA repair activity in vivo and in vitro. The expression and activity of DNA repair enzymes will be studied and the effect of pharmacologic inhibition of these enzymes upon DNA damage and neuronal survival investigated.